In individuals with sickle cell trait, i.e. the heterozygous state for sickle cell anemia, the peripheral blood concentration of the abnormal hemoglobin S (HbS) is lower than that of the normal hemoglobin A (HbA). The reasons for this difference are largely unknown; it may occur because of decreased systhesis and/or enhanced degradation of HbS compared to that of HbA. The major objective is to determine the molecular basis for the blood concentration disparity of Hbs A and S by studying patterns of hemoglobin synthesis. The long term goal is to clarify the mechanisms of transcriptional and translational control in cells and tissues in general and possibly to use the knowledge learned in the treatment of individuals with inherited disorders. In preliminary work a method was developed to obtain from moderate amounts of sickle cell trait blood a fraction of cells (15 to 25% reticulocytes) actively synthesizing hemoglobin. Using these cells a few sickle cell trait donors with low concentrations of HbS were found to carry a gene for alpha-thalassemia, a disease characterized by a deficit in hemoglobin alpha chain synthesis. The specific aims of the proposed project are: (a) to determine if all, or nearly all, of these individuals with low HbS concentrations carry an alpha-thalassemia gene, (b) to determine the detailed intracellular patterns of HbA and HbS synthesis and assembly from individual globin chains in reticulocytes of high and low HbS donors, and (c) to determine if a putative relative decrease in Beta chain synthesis occurs because of a decrease in amount of messenger RNA or, conversely, a decrease in messenger RNA translational activity. Reticulocytes will be incubated with H3 leucine and lysed. Various cell fractions, including the supernatant and stroma, will be isolated, and Hbs A and S or the globin chains will be separated and analyzed for concentration and radioactivity by two major procedures: (a) electrophoresis on strips of cellulose acetate, and (b) column chromatography on CM-cellulose to separate the globin chains. The rates of synthesis and turnover of the two hemoglobins and their individual chains will be used to determine the basis for the disparity in the blood concentrations of Hbs A and S.